The Cell Polarity Protein mInsc Regulates Neutrophil Chemotaxis via a Noncanonical G Protein Signaling Pathway

Abstract

Successful chemotaxis requires not only increased motility but also sustained directionality. Here, we show that, during neutrophil chemotaxis via receptors coupled with the Gi family of heterotrimeric G proteins, directional movement is regulated by mInsc, a mammalian protein distantly related to the Drosophila polarity-organizer Inscuteable. The GDP-bound, Gβγ-free Gαi subunit accumulates at the front of chemotaxing neutrophils to recruit mInsc-complexed with the Par3-aPKC evolutionarily conserved polarity complex-via LGN/AGS3 that simultaneously binds to Gαi-GDP and mInsc. Both mInsc-deficient and aPKC-blocked neutrophils exhibit a normal motile activity but migrate in an undirected manner. mInsc deficiency prevents neutrophils from efficiently stabilizing pseudopods at the leading edge; the stability is restored by wild-type mInsc, but not by a mutant protein defective in binding to LGN/AGS3. Thus, mInsc controls directional migration via noncanonical G protein signaling, in which Gβγ-free Gαi-GDP, a product from Gαi-GTP released after receptor activation, plays a central role.

Activators of G Protein Signaling Exhibit Broad Functionality and Define a Distinct Core Signaling Triad

“…GIV is also localized to the centrosome and the midbody as observed for AGS5 (Blumer et al, 2006;Mao et al, 2012). Interestingly, group II AGS proteins AGS5/ LGN and AGS3 are also able to interact with and influence the subcellular distribution of the PAR complex to regulate cell polarity and in some cases orient the mitotic spindle (Lechler and Fuchs, 2005;Zigman et al, 2005;Izaki et al, 2006;Yuzawa et al, 2011;Kamakura et al, 2013;). As Gai-AGS3 is a substrate for the GEF activity of GIV (Garcia-Marcos et al, 2011a), this may illustrate yet another area of possible connectivity between group I and group II AGS proteins.…”

“…1). In mammalian systems, AGS3 is implicated in asymmetric cell division, neuronal plasticity and addiction, autophagy, membrane protein trafficking, polycystic kidney disease, renal response to ischemia, immune cell chemotaxis, insulin-like growth factor-1 mediated ciliary resorption, cardiovascular regulation, and metabolism (Blumer et al, 2002(Blumer et al, , 2006(Blumer et al, , 2008Ghosh et al, 2003;Gotta et al, 2003;Pattingre et al, 2003;Bowers et al, 2004Bowers et al, , 2008Sato et al, 2004;Yao et al, 2005Yao et al, , 2006Groves et al, 2007;Willard et al, 2008;Groves et al, 2010;Nadella et al, 2010;Vural et al, 2010;Hofler and Koelle, 2011;Regner et al, 2011;Chauhan et al, 2012;Kwon et al, 2012;Conley and Watts, 2013;Kamakura et al, 2013;Yeh et al, 2013). AGS3 single nucleotide polymorphisms are associated with diabetes and glucose handling (Scott et al, 2012;Hara et al, 2014;Huyghe et al, 2013).…”

“…In addition, the newly generated Ga-GPR may initiate the formation of a signaling complex that acts independently of Gbg-mediated signaling. For example, during neutrophil chemotaxis the GaiAGS3 complex acts as a docking site for polarized formation of a mInsc-Par3-Par6-aPKC complex, which is important for efficient chemokine-directed migration (Kamakura et al, 2013). Such a GaGPR complex could also serve as a target for group I AGS proteins, as suggested for signaling events associated with feeding behavior in C. elegans (Hofler and Koelle, 2011).…”

The Nuclear Protein IκBζ Forms a Transcriptionally Active Complex with Nuclear Factor-κB (NF-κB) p50 and the Lcn2 Promoter via the N- and C-terminal Ankyrin Repeat Motifs

“…GST Pulldown Assay-GST-and His-tagged proteins were purified as described previously (52,53) and incubated for 20 min at 4°C in 500 l of buffer A (137 mM NaCl, 2.7 mM KCl, 0.5% Triton X-100, 1 mM DTT, 8.1 mM Na 2 HPO 4 , and 1.5 mM KH 2 PO 4 (pH 7.4)). A slurry of glutathione-Sepharose 4B beads (GE Healthcare) was subsequently added, followed by further incubation for 40 min at 4°C.…”

Structural basis for the recognition of the scaffold protein Frmpd4/Preso1 by the TPR domain of the adaptor protein LGN

“…The adaptor protein LGN plays a crucial role in mitotic spindle orientation and cell polarization via interaction with multiple targets such as the nuclear mitotic apparatus protein NuMA and the cell-polarity protein mInsc (Du & Macara, 2004;Ž igman et al, 2005;Konno et al, 2008;Kamakura et al, 2013;Williams et al, 2014). The N-terminal half of LGN comprises eight copies of a tetratricopeptide-repeat (TPR) motif, each adopting a helix-turn-helix structure of A and B , to form a right-handed superhelical domain (Yuzawa et al, 2011;Zhu et al, 2011), whereas the C-terminal half contains four GoLoco/GPR motifs, each capable of binding to GDPbound G i (Willard et al, 2004).…”